CN105420324A - Preparation method of high-molecular-weight silver carp skin collagen peptide - Google Patents
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Abstract
The invention relates to a preparation method of high-molecular-weight silver carp skin collagen peptide. The method includes the following steps of firstly, cleaning fresh silver carp skin to remove impurities, conducting degreasing through an alkali solution, removing impurity protein through an acid solution, and conducting rinsing to be nearly neutral; secondly, adding water to preprocessed silver carp skin, adjusting pH to be 3.5 to 4.5 and the temperature to be 40 DEG C to 45 DEG C, adding pepsase for an enzymatic hydrolysis reaction for the reaction time of 10 h to18 h, and keeping the pH and the temperature constant in the reaction process; thirdly, conducting ultra-low-temperature vacuum concentration on enzymatic hydrolysate of pepsase; fourthly, controlling the reaction conditions for a protein transfer reaction to obtain a large-molecule collagen peptide solution, and then conducting processing on the collagen peptide solution. By means of the method, protein synthesis is conducted on the basis of protein hydrolysis through the protein transfer reaction under the control conditions, large-molecule collagen peptide is obtained, molecular weight distribution is concentrated, raw materials and auxiliary materials are high in utilization rate, the process is simple, and processing cost is low.
Description
Technical field
The present invention relates to protein peptide preparation field in biotechnology, be specifically related to a kind of method with turning albumen test and prepare high molecular Silver Carp collagen peptide.
Background technology
Collagen protein, extremely important a kind of structural protein in reticular tissue, the protein that animal body intensive amount is maximum, distribution is the widest, account for 25% ~ 30% of body internal protein total amount, and the excessive collagen protein of molecular weight is low at the absorption rate of human body, need through the collagen peptide of biotechnology degraded to molecular weight to 200 ~ 10000Da scope, human body could effectively absorb.Research proves that the molecular-weight average of protein hydrolystate determines one of most important factor of its biological property, and physics and chemistry, the functional performance of protein peptide component are relevant with its molecular size range.
The application of being extracted collagen peptide by pigskin, ox-hide is comparatively extensive, but fish-skin is cheap, abundance and extraction comparison easy, and without the risk of terrestrial animal epidemic disease.China is freshwater fish culturing state maximum in the world, along with the fast development of freshwater fish processing industry, a large amount of fish-skins and fish scale the like waste is produced in the course of processing, as insufficient utilization, also great waste is caused to resource while contaminate environment, therefore study and from fish scrap, extract collagen peptide there is far reaching significance.
Chinese patent application publication No. CN102621213A discloses a kind of evaluation method of acidity pepsin method macromolecular collagen protein thermally denature degree, obtain the complete triple-helix structure collagen protein of molecular weight at 300,000 more than Da by after raw material soaking dissolving, heating, enzymolysis, and the thermally denature degree of collagen protein is evaluated.Chinese patent CN102676620B discloses a kind of preparation method of high-molecular-weighsquid squid skin collagen, selects molecular weight cut-off to be that the ultra-filtration membrane of 100kda carries out ultrafiltration to enzymolysis solution, reclaims micromolecular collagen peptide while obtaining macromolecular collagen protein peptide.Chinese patent application publication number 103627763A discloses a kind of stage division of anglerfish skin collagen protein peptide molecular weight, anglerfish skin enzymolysis solution is through the collagen peptide of one-level plate-type hyperfiltration retaining molecular weight more than 10000 dalton, then through the collagen peptide of secondary spiral wound retaining molecular weight more than 2000 dalton, more afterwards through the collagen peptide of rolling nanofiltration membrane molecular weight cut-off more than 200 dalton.Between collagen peptide prepared by above method, molecular size range gap is very far away, and distribution is not concentrated, and obtains the collagen peptide of different molecular weight section eventually through ultrafiltration classification, complex process and cost is higher.
Summary of the invention
Goal of the invention of the present invention is that the deficiency existed for prior art provides a kind of method preparing high molecular Silver Carp collagen peptide.The method is by turning the macromolecular collagen protein peptide that synthesis that albumen test carries out protein again on the basis of proteolysis obtains under control condition, and molecular weight distribution is concentrated, and supplementary material utilization ratio is high, and technique is simple, and tooling cost is low.
The object of the invention is to be achieved by following technical solution:
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1), after foreign material being removed in the cleaning of fresh white silver carp skin, first use alkaline solution degreasing, then remove foreign protein with acid solution, rinsing is to weakly acidic pH (pH6.5-7.5);
(2) added water by silver carp fish-skin good for pre-treatment, regulate pH to 3.5 ~ 4.5, temperature to 40 ~ 45 DEG C, add stomach en-and carry out enzyme digestion reaction, reaction times 10 ~ 18h, maintain pH and homo(io)thermism in reaction process;
(3) carry out ultra low temperature vacuum to above-mentioned stomach en-enzymolysis solution to concentrate;
(4) control reaction conditions to carry out turning egg(s) and be obtained by reacting macromolecular collagen protein peptide solution in vain, aftertreatment secures satisfactory grades son amount Silver Carp collagen peptide.
Further, described degreasing, for soak 6-10h degreasing with 0.2% ~ 0.4% alkaline solution (w:v, g/ml), is rinsed to weakly acidic pH; The described foreign protein that removes removes foreign protein for soaking 3 ~ 6h with 0.1% ~ 0.3% acid solution (w:v, g/ml); First wash after described degreasing to weakly acidic pH and remove foreign protein again.
Further, the amount of degreasing alkaline solution used is 4 ~ 6 times of starting raw material fresh white silver carp tare weight amount, and the amount removing foreign protein acid solution used is 4 ~ 6 times of starting raw material fresh white silver carp tare weight amount.
Further, described alkali is one or more the mixing in sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and described acid is one or more the mixing in hydrochloric acid, nitric acid, phosphoric acid, citric acid, oxalic acid.
Further, in step (2), amount of water is starting raw material fresh white silver carp cortex amount 1.5 ~ 2.5 times, and pepsic consumption is 0.1 ~ 0.3% of starting raw material fresh white silver carp cortex amount.
Further, in step (3), very low temperature thickening temperature controls at 30 ~ 45 DEG C, and final concentrated solution mass percent concentration controls 30% ~ 35%.
Further, regulate reaction system pH6.0 ~ 6.5 in step (4), temperature 40 ~ 45 DEG C, carries out turning albumen test, reaction times 1 ~ 2h.
Further, the whole ultra low temperature vacuum concentration process in step (3) carries out under without shearing force or low-shearing power operating mode, to retain pepsic activity.As when ultra low temperature vacuum concentrates, ultra low temperature vacuum thickening equipment is supported the use the sine pump without shearing force, ensure that whole ultra low temperature vacuum concentration process carries out under without shearing force operating mode, to retain pepsic activity.
Further, described aftertreatment for obtaining collagen protein Gly-His-Lys by white for turning egg(s) reaction solution after the enzyme that goes out, decoloration and deodorization, Sterile Filtration, spraying dry.
Further, enzyme-removal temperature 85 ~ 90 DEG C, time 10 ~ 20min.
Further, decoloration and deodorization gac addition be reaction solution 1% ~ 3%(w:v, g/ml), temperature 60 ~ 70 DEG C, time 30 ~ 60min.
Further, Sterile Filtration adopts 0.45 μm of+0.22 μm of combined filter, and destainer is filtered to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering.
Further, spraying dry inlet temperature 190 ~ 200 DEG C, goes out inlet temperature 90 ~ 95 DEG C, atomizing pressure 40 ~ 60bar.
compared with prior art, advantage of the present invention and beneficial effect are:
1, the present invention by obtaining macromolecular collagen protein peptide by the synthesis that albumen test carries out protein that turns under control condition on the basis of proteolysis, and molecular weight distribution is concentrated, and without the need to follow-up stage treatment, raw material availability is high;
2, the decomposition of protein and synthesis use the same enzyme to complete (only need change, control reaction conditions), and cost is low.
Embodiment
Applicant will be further elaborated the inventive method in conjunction with specific embodiments below, should be understood that following examples only for describing technical scheme of the present invention in detail, and should not be understood to the restriction to request protection domain of the present invention in any degree.
embodiment 1
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1) take the fresh silver carp fish-skin of 1000Kg, rinse and remove foreign matter, with the 0.2% soaking in sodium carbonate solution 8h that 4 times of fish-skins are heavy, rinse to weakly acidic pH, then with the heavy 0.1% hydrochloric acid soln immersion 3h of 5 times of fish-skins, rinse to weakly acidic pH;
(2) washed fish-skin is dropped into retort, add 2000Kg pure water, open and stir, mixture pH to 4.5 is regulated with dilute hydrochloric acid, lead to steam to retort chuck and be warming up to about 42 DEG C, thing temperature to be mixed and pH constant after add 2Kg stomach en-, maintain initial pH and temperature stirring reaction 12h;
(3) with ultra low temperature vacuum thickening equipment, enzymolysis solution is concentrated, thickening temperature 40 DEG C, when concentrated solution mass percent concentration reaches 34%, go to retort, the recycle pump that wherein ultra low temperature vacuum thickening equipment uses is the sine pump without shearing force, fully to retain pepsic activity;
(4) regulate concentrated solution pH to 6.0, temperature 40 DEG C, stir and carry out turning albumen test 1.5h;
(5) white for turning egg(s) reaction solution be warming up to 87 DEG C and proceed in bleacher after maintaining 10min, cooling water temperature to 68 DEG C is led to bleacher chuck, add reaction solution 1%(w:v again, g/ml) Powdered Activated Carbon stirs decolouring 30min, destainer filters to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering, and bacteria-free filtrate spraying dry under inlet temperature 195 DEG C, air outlet temperature 93 DEG C, atomizing pressure 45bar condition obtains collagen protein Gly-His-Lys 180Kg.
Gained collagen peptide moisture 4.8%, ash content 0.2%, total nitrogen 16.7%, molecular weight occupy and account for 70%, free aminoacid content 1.65% between 1638Da and 5808Da after testing.
embodiment 2
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1) take the fresh silver carp fish-skin of 2000Kg, rinse and remove foreign matter, 0.3% sodium hydroxide solution heavy with 6 times of fish-skins soaks 6h, rinses to weakly acidic pH, then with the heavy 0.2% citric acid solution immersion 6h of 5 times of fish-skins, rinses to weakly acidic pH;
(2) washed fish-skin is dropped into retort, add 3000Kg pure water, open and stir, mixture pH to 4.4 is regulated with citric acid, lead to steam to retort chuck and be warming up to about 45 DEG C, thing temperature to be mixed and pH constant after add 3Kg stomach en-, maintain initial pH and temperature stirring reaction 15h;
(3) with ultra low temperature vacuum thickening equipment, enzymolysis solution is concentrated, thickening temperature 45 DEG C, when concentrated solution mass percent concentration reaches 32%, go to retort, the peristaltic pump that the recycle pump that wherein ultra low temperature vacuum thickening equipment uses is low-shearing power, fully to retain pepsic activity;
(4) regulate concentrated solution pH to 6.2, temperature 42 DEG C, stir and carry out turning albumen test 1h;
(5) white for turning egg(s) reaction solution be warming up to 85 DEG C and proceed in bleacher after maintaining 15min, cooling water temperature to 70 DEG C is led to bleacher chuck, add reaction solution 1.5%(w:v again, g/ml) Powdered Activated Carbon stirs decolouring 45min, destainer filters to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering, and bacteria-free filtrate spraying dry under inlet temperature 190 DEG C, air outlet temperature 95 DEG C, atomizing pressure 42bar condition obtains collagen protein Gly-His-Lys 350Kg.
Gained collagen peptide moisture 4.2%, ash content 0.15%, total nitrogen 16.8%, molecular weight occupy and account for 72%, free aminoacid content 1.38% between 1638Da and 5808Da after testing.
embodiment 3
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1) take the fresh silver carp fish-skin of 2500Kg, rinse and remove foreign matter, 0.4% aqua calcis heavy with 5 times of fish-skins soaks 10h, rinses to weakly acidic pH, then with the heavy 0.1% salpeter solution immersion 4h of 4 times of fish-skins, rinses to weakly acidic pH;
(2) washed fish-skin is dropped into retort, add 4000Kg pure water, open and stir, mixture pH to 3.5 is regulated with dilute hydrochloric acid, lead to steam to retort chuck and be warming up to about 41 DEG C, thing temperature to be mixed and pH constant after add 2.5Kg stomach en-, maintain initial pH and temperature stirring reaction 18h;
(3) with ultra low temperature vacuum thickening equipment, enzymolysis solution is concentrated, thickening temperature 44 DEG C, when concentrated solution mass percent concentration reaches 35%, go to retort, the spiral pump that the recycle pump that wherein ultra low temperature vacuum thickening equipment uses is low-shearing power, fully to retain pepsic activity;
(4) regulate concentrated solution pH to 6.5, temperature 42 DEG C, stir and carry out turning albumen test 2h;
(5) white for turning egg(s) reaction solution be warming up to 90 DEG C and proceed in bleacher after maintaining 10min, cooling water temperature to 60 DEG C is led to bleacher chuck, add reaction solution 2%(w:v again, g/ml) Powdered Activated Carbon stirs decolouring 60min, destainer filters to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering, and bacteria-free filtrate spraying dry under inlet temperature 200 DEG C, air outlet temperature 90 DEG C, atomizing pressure 60bar condition obtains collagen protein Gly-His-Lys 500Kg.
Gained collagen peptide moisture 4.5%, ash content 0.1%, total nitrogen 16.3%, molecular weight occupy and account for 68%, free aminoacid content 1.25% between 1638Da and 5808Da after testing.
embodiment 4
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1) take the fresh silver carp fish-skin of 1500Kg, rinse and remove foreign matter, 0.3% aqua calcis heavy with 5.5 times of fish-skins soaks 7h, rinses to weakly acidic pH, then with the heavy 0.3% citric acid solution immersion 5h of 6 times of fish-skins, rinses to weakly acidic pH;
(2) washed fish-skin is dropped into retort, add 3750Kg pure water, open and stir, mixture pH to 3.8 is regulated with dilute hydrochloric acid, lead to steam to retort chuck and be warming up to about 40 DEG C, thing temperature to be mixed and pH constant after add 4.5Kg stomach en-, maintain initial pH and temperature stirring reaction 10h;
(3) with ultra low temperature vacuum thickening equipment, enzymolysis solution is concentrated, thickening temperature 30 DEG C, when concentrated solution mass percent concentration reaches 30%, go to retort, the rotor pump that the recycle pump that wherein ultra low temperature vacuum thickening equipment uses is low-shearing power, fully to retain pepsic activity;
(4) regulate concentrated solution pH to 6.4, temperature 45 C, stir and carry out turning albumen test 1.5h;
(5) white for turning egg(s) reaction solution be warming up to 88 DEG C and proceed in bleacher after maintaining 20min, cooling water temperature to 67 DEG C is led to bleacher chuck, add reaction solution 2.5%(w:v again, g/ml) Powdered Activated Carbon stirs decolouring 50min, destainer filters to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering, and bacteria-free filtrate spraying dry under inlet temperature 192 DEG C, air outlet temperature 94 DEG C, atomizing pressure 40bar condition obtains collagen protein Gly-His-Lys 315Kg.
Gained collagen peptide moisture 4.7%, ash content 0.12%, total nitrogen 16.6%, molecular weight occupy and account for 63%, free aminoacid content 1.47% between 1638Da and 5808Da after testing.
embodiment 5
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1) take the fresh silver carp fish-skin of 1800Kg, rinse and remove foreign matter, 0.4% sodium hydrogen carbonate solution heavy with 4.5 times of fish-skins soaks 9h, rinses to weakly acidic pH, then with the heavy 0.2% oxalic acid solution immersion 5.5h of 6 times of fish-skins, rinses to weakly acidic pH;
(2) washed fish-skin is dropped into retort, add 3500Kg pure water, open and stir, mixture pH to 4.1 is regulated with dilute hydrochloric acid, lead to steam to retort chuck and be warming up to about 43 DEG C, thing temperature to be mixed and pH constant after add 2.7Kg stomach en-, maintain initial pH and temperature stirring reaction 12h;
(3) with ultra low temperature vacuum thickening equipment, enzymolysis solution is concentrated, thickening temperature 33 DEG C, when concentrated solution mass percent concentration reaches 30%, go to retort;
(4) regulate concentrated solution pH to 6.1, temperature 43 DEG C, stir and carry out turning albumen test 1h;
(5) white for turning egg(s) reaction solution be warming up to 87 DEG C and proceed in bleacher after maintaining 10min, cooling water temperature to 65 DEG C is led to bleacher chuck, add reaction solution 3%(w:v again, g/ml) Powdered Activated Carbon stirs decolouring 30min, destainer filters to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering, and bacteria-free filtrate spraying dry under inlet temperature 198 DEG C, air outlet temperature 92 DEG C, atomizing pressure 45bar condition obtains collagen protein Gly-His-Lys 358Kg.
Gained collagen peptide moisture 4.9%, ash content 0.23%, total nitrogen 16.7%, molecular weight occupy and account for 65%, free aminoacid content 1.38% between 1638Da and 5808Da after testing.
embodiment 6
Prepare a method for high molecular Silver Carp collagen peptide, its step is as follows:
(1) take the fresh silver carp fish-skin of 2200Kg, rinse and remove foreign matter, 0.2% potassium hydroxide solution heavy with 6 times of fish-skins soaks 7h, rinses to weakly acidic pH, then with the heavy 0.15% hydrochloric acid soln immersion 3.5h of 4 times of fish-skins, rinses to weakly acidic pH;
(2) washed fish-skin is dropped into retort, add 3300Kg pure water, open and stir, mixture pH to 4.3 is regulated with dilute hydrochloric acid, lead to steam to retort chuck and be warming up to about 44 DEG C, thing temperature to be mixed and pH constant after add 2.2Kg stomach en-, maintain initial pH and temperature stirring reaction 17h;
(3) with ultra low temperature vacuum thickening equipment, enzymolysis solution is concentrated, thickening temperature 38 DEG C, when concentrated solution mass percent concentration reaches 34%, go to retort;
(4) regulate concentrated solution pH to 6.4, temperature 41 DEG C, stir and carry out turning albumen test 1.2h;
(5) white for turning egg(s) reaction solution be warming up to 85 DEG C and proceed in bleacher after maintaining 20min, cooling water temperature to 63 DEG C is led to bleacher chuck, add reaction solution 1%(w:v again, g/ml) Powdered Activated Carbon stirs decolouring 60min, destainer filters to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering, and bacteria-free filtrate spraying dry under inlet temperature 193 DEG C, air outlet temperature 90 DEG C, atomizing pressure 53bar condition obtains collagen protein Gly-His-Lys 407Kg.
Gained collagen peptide moisture 4.1%, ash content 0.35%, total nitrogen 16.4%, molecular weight occupy and account for 67%, free aminoacid content 1.79% between 1638Da and 5808Da after testing.
Claims (10)
1. prepare a method for high molecular Silver Carp collagen peptide, it is characterized in that: step is as follows:
(1), after foreign material being removed in the cleaning of fresh white silver carp skin, first use alkaline solution degreasing, then remove foreign protein with acid solution, rinsing is to weakly acidic pH;
(2) added water by silver carp fish-skin good for pre-treatment, regulate pH to 3.5 ~ 4.5, temperature to 40 ~ 45 DEG C, add stomach en-and carry out enzyme digestion reaction, reaction times 10 ~ 18h, maintain pH and homo(io)thermism in reaction process;
(3) carry out ultra low temperature vacuum to above-mentioned stomach en-enzymolysis solution to concentrate;
(4) control reaction conditions to carry out turning egg(s) and be obtained by reacting macromolecular collagen protein peptide solution in vain, aftertreatment secures satisfactory grades son amount Silver Carp collagen peptide.
2. the method preparing high molecular Silver Carp collagen peptide according to claim 1, is characterized in that: described degreasing, for soak 6-10h degreasing with 0.2% ~ 0.4% alkaline solution (w:v, g/ml), is rinsed to weakly acidic pH; The described foreign protein that removes removes foreign protein for soaking 3 ~ 6h with 0.1% ~ 0.3% acid solution (w:v, g/ml); After described degreasing, subpunch is washed till neutrality and removes foreign protein again.
3. the method preparing high molecular Silver Carp collagen peptide according to claim 2, it is characterized in that: the amount of degreasing alkaline solution used is 4 ~ 6 times of starting raw material fresh white silver carp tare weight amount, the amount removing foreign protein acid solution used is 4 ~ 6 times of starting raw material fresh white silver carp tare weight amount.
4. the method preparing high molecular Silver Carp collagen peptide according to claim 1 and 2, it is characterized in that: alkali is one or more the mixing in sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, acid is one or more the mixing in hydrochloric acid, nitric acid, phosphoric acid, citric acid, oxalic acid.
5. the method preparing high molecular Silver Carp collagen peptide according to claim 1, it is characterized in that: in step (2), amount of water is starting raw material fresh white silver carp cortex amount 1.5 ~ 2.5 times, and pepsic consumption is 0.1 ~ 0.3% of starting raw material fresh white silver carp cortex amount.
6. the method preparing high molecular Silver Carp collagen peptide according to claim 1, is characterized in that: in step (3), very low temperature thickening temperature controls at 30 ~ 45 DEG C, and final concentrated solution mass percent concentration controls 30% ~ 35%.
7. the method preparing high molecular Silver Carp collagen peptide according to claim 1, is characterized in that: regulate reaction system pH6.0 ~ 6.5 in step (4), temperature 40 ~ 45 DEG C, carries out turning albumen test, reaction times 1 ~ 2h.
8. the method preparing high molecular Silver Carp collagen peptide according to claim 1, is characterized in that: the whole ultra low temperature vacuum concentration process in step (3) carries out under without shearing force or low-shearing power operating mode, to retain pepsic activity.
9. the method preparing high molecular Silver Carp collagen peptide according to claim 1, is characterized in that: described aftertreatment for obtaining collagen protein Gly-His-Lys by white for turning egg(s) reaction solution after the enzyme that goes out, decoloration and deodorization, Sterile Filtration, spraying dry.
10. the method preparing high molecular Silver Carp collagen peptide according to claim 1, is characterized in that:
Enzyme-removal temperature 85 ~ 90 DEG C, time 10 ~ 20min;
Decoloration and deodorization gac addition be reaction solution 1% ~ 3%(w:v, g/ml), temperature 60 ~ 70 DEG C, time 30 ~ 60min;
Sterile Filtration adopts 0.45 μm of+0.22 μm of combined filter, and destainer is filtered to obtain bacteria-free filtrate through 0.22 μm of accurate filter again after 0.45 μm of strainer pre-filtering;
Spraying dry inlet temperature 190 ~ 200 DEG C, goes out inlet temperature 90 ~ 95 DEG C, atomizing pressure 40 ~ 60bar.
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| CN114106152A (en) * | 2022-01-26 | 2022-03-01 | 广州创尔生物技术股份有限公司 | Method for preparing collagen with triple-helical structure by spray drying |
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| CN105420324B (en) | 2019-02-15 |
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